During the previous funding periods of this grant, we learned fundamental aspects of the mechanism by which the protozoan parasite Trypanosoma cruzi gains access to the intracellular environment. Host cell lysosomes play a key role in this process. Trypomastigotes actively invade non-phagocytic cells by one of two mechanisms: 1) triggering Ca2+ signaling events at the surface of host cells that lead to lysosomal recruitment and exocytosis, a process that delivers intracellular membrane for formation of the parasitophorous vacuole; 2) by invaginating the plasma membrane, a process that is rapidly followed by lysosomal fusion. Our studies revealed that in the absence of lysosomal fusion the T. cruzi invasion process is reversible, with infective trypomastigotes being released back into the extracellular medium. Thus, regardless of whether the initial cell entry event involves lysosomal fusion with the plasma membrane ornot, lysosomal fusion is essential to retain the parasites inside host cells, allowing completion of the life cycle. In the next funding period, we plan to test the hypothesis that lysosomal fusion is critical for T. cruzi invasion in two ways: 1) by delivering a high affinity trypomastigote receptor to the host cell surface, and 2) by acting as an intracellular anchor for trypomastigotes by promoting strong association to host cell microtubules. In AIM 1 we will investigate whether the T. cruzi transialidase binds sialic acid moieties on the luminal domain of lysosomal glycoproteins, ensuring the close apposition of parasite and host cell membranes during invasion. To this end,in addition to biochemical interaction assays we will examine parasites expressingdifferent levels of transialidase, and host cell fibroblasts from mice deficient in Lampl and Lamp2, the two major sialic acid-containing glycoproteins of mammalian cell lysosomes. In AIM 2 we will examine the mechanisms by which lysosomal fusion and host cell microtubules retain T. cruzi trypomastigotes inside host cells. Our hypothesis is that this process is mediated by specific molecular motors that link lysosomes to microtubules. In AIM 3, we will investigate whether lysosomal proteases play an important role in the escape of T. cruzi from the vacuole where it resides shortly after infection. As in the past, we are confident that the new knowledge that we will gain in this process will advance our understanding of this important human parasite, and also uncover novel aspects of the biology of mammalian cells.